Congenital obstructive nephropathy is the most frequent cause of renal failure in infants and children, and if left untreated can cause severe renal failure and death. Challenges in the evaluation and management include the lack of clear indications if and when surgical intervention is indicated, the use of controversial invasive diagnostic tests that may employ radiation, and the requirement for long-term follow-up to monitor disease progression that often occurs despite surgical correction. The goal of this proposal is to develop a sensitive multiplex assay of urinary biomarkers of renal inflammation and cell injury to aid the diagnosis, prognosis, therapeutic decision-making, and monitoring of patient therapy. We have developed a proprietary technology using the real-time-polymerase chain reaction (PCR) technology to measure protein concentrations in a multiplexed fashion with very high sensitivity and reproducibility called Multiplexed Real-time-Immuno-PCR (MRI-PCR). In Phase I studies, a panel of reagents to detect human biomarkers of obstructive nephropathy will be prepared and evaluated in the MRI-PCR assay. A comprehensive analytical validation of the MRI-PCR assay will be performed by measuring sensitivity, specificity, accuracy, and dilutional linearity of each analyte. The performance will be verified by spiking known concentrations of each of the analytes into normal urine. In Phase II, the assay will be validated by testing urine samples from patients with congenital urinary tract obstruction as well as urine from healthy children. The successful completion of this project will result in an assay of multiple urinary biomarkers to aid clinicians and researchers in the evaluation and management of congenital obstructive nephropathy. Although the initial focus is on neonatal and childhood urinary tract obstructions, ultimately this assay may be useful for adults with obstructed nephropathy and perhaps other types of renal diseases. Congenital obstructive nephropathy is a common disease in neonate that is difficult to clinically assess without the use of invasive imaging modalities. This proposal will develop a non-invasive new technology that will simultaneously measure a panel of urinary biomarkers associated with obstructed urinary tract diseases that is expected to be useful for diagnosis, prognosis, guiding therapeutic intervention, and long-term monitoring of responses to therapy or disease progression. [unreadable] [unreadable] [unreadable]